In vitro evaluation on the anti-apoptotic and anti-oxidant effect of a series of natural polyphenols on UVB- irradiated normal human keratinocytes and melanocytes
UV radiation, in particular its UVB component, is an important environmental factor in the pathogenesis of skin aging and cancer.One of the features of UVB-caused DNA damage is the formation of cyclobutane pyrimidine dimers and (6-4) photoproducts. Further, indirect DNA damage is also caused by increase in the level of reactive oxygen species (ROS) (1) that cause oxidative damage reacting with DNA, proteins, fatty acids and saccharides (2). Such injuries result in a number of harmful effects: disturbed cell metabolism, morphological and ultrastructural changes, attack on the regulation pathways and, alterations in the differentiation, proliferation and apoptosis of skin cells (3). These processes can lead to photoaging and skin cancer development. In order to avoid UVB radiation damage, phytocompounds and antioxidants as photoprotectives has been considered (4). In recent years naturally occurring herbal compounds such as phenolic acids, flavonoids, and high molecular weight polyphenols have gained remarkable attention as strong protective agents (5,6). In the present study we considered a series of natural polyphenols with the aim to investigate on their anti-oxidant and anti-apoptotic effect in UVB-irradiated normal human keratinocytes and melanocytes.Methods: growing concentration (from 10 to 100 M) of forty-six polyphenols selected by the Department of Pharmaceutical Sciences of the University of Modena were added to normal human keratinocytes and melanocytes coltures. In the first part of the study we assessed the activity on UVB-irradiated normal human keratinocytes and melanocytes. These substances were submitted to a primary in vitro screening by MTT test in order to valuate proliferation rate. Subsequently, 2’7’- dichlorodihydrofluorescein diacetate (DCF) assay was performed to determine the formation of intracellular ROS. The results showed that glabridin, 18β-glycyrrhetinic acid, kaempferol, quercetin, rutin and butein presented interesting properties and a low toxicity. In the second part of the study the antiapoptotic effects of these six selected compounds in UVB-irradiated (50 mJ/cm2/11’and 44’’) normal human keratinocytes and melanocytes was investigated by the western blot analysis.Results: with regard to analysed polyphenols, the results showed that a pre-treatment of human keratinocytes and melanocytes with these molecules inhibited UVB mediated apoptosis through the activation of p53, down-regulation of bcl-2 and bid full-length, up- regulation of bax and inhibition of PARP cleavage. For these purpose we proposed that some substances could be combined to evaluate their synergistic effects.Conclusion: To further elucidate the molecular mechanism of polyphenol or more generally phytocompounds mediated apoptosis, we decided to investigate cell cycle analysis and the extrinsic and intrinsic apoptotic pathways. Human skin is constantly exposed to the UV radiation present in sunlight. This may induce a number of pathobiological cellular changes. The development of novel preventive and therapeutic strategies depends on our understanding of the molecular mechanism of UV-damage. Phytochemical that were identified may be candidates for prevention of adverse effects of UV radiation and melanoma on the skin and evaluation of there clinical efficacy is awaited
